Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Summary

Accurately quantifying Salmonella in poultry at low levels is a current industrial and regulatory challenge. This protocol describes an MPN assay that enables quantification of Salmonella in raw and ready-to-cook poultry products. This method is fast, sensitive, and aligns with FSIS guidelines, enhancing food safety and supporting public health efforts.

Abstract

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R² = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Introduction

As a leading cause of foodborne illness, hospitalization, and death in the U.S., Salmonella has a significant public health and economic impact. The pathogen's estimated economic burden in 2013 alone was $3.67 billion¹. Although recent regulatory initiatives aim to reduce salmonellosis by 25% by 2030², gaps in current detection and mitigation strategies remain evident, particularly in aligning processing plant surveillance with public health outcomes³ .

Frozen ready-to-cook poultry products, which have been implicated in multiple Salmonella outbreaks, are a significant concern for public health. In response, the Food Safety and Inspection Service (FSIS) classified Salmonella as an adulterant in these products. Currently, FSIS Microbiology Laboratory Guidebook (MLG) 4.15 focuses solely on determining the prevalence of Salmonella in poultry products⁴. Under this guideline, collected samples are enriched for 18-24 h and then screened using the Molecular Detection System (MDS), which identifies the presence or absence of Salmonella but does not offer insight into the level of contamination. While this approach is valuable for detecting the presence of pathogens, it fails to provide quantitative information that could help food processors assess contamination risks more accurately and take targeted corrective actions.

In this study, we developed a method to augment detection from prevalence to quantification of microbial pathogens. It was designed for seamless integration into existing processes to detect Salmonella in poultry products with minimal disruption to current FSIS protocols. Instead of simply enriching the bulk sample, the method begins by washing the poultry products using media consistent with current FSIS methods. The rinse is then distributed into the first column of a 48-deep well block. Serial dilutions are performed across the remaining five columns, and the block is incubated for 18-24 h, aligning with the MLG 4.15 protocol. After incubation, the wells are tested for Salmonella, and the results are used to calculate the most probable number (MPN)^5,6. This approach allows for quantification of contamination within the same time frame as the current FSIS process, making it a practical option for both industry and regulatory use. Figure 1 depicts a block diagram summarizing the modified MPN assay. The figure includes photographs taken at specific steps, the 48-well block utilized for dilution and growth of replicates, and the three techniques used as benchmarks to assess the most probable number of Salmonella present in ground chicken. In the first phase of this study, we utilized irradiated ground chicken to minimize the impact of background microflora and uncertainty of measurements relative to verified inoculum before applying the protocol to non-irradiated chicken samples.

Protocol

NOTE: All work associated with this protocol should be conducted within a Biosafety Level 2 (BSL-2) laboratory. When appropriate, this protocol should be conducted within a biological safety cabinet (BSC) to maintain aseptic conditions and minimize the risk of sample contamination or operator exposure to microbial pathogens. When transferring samples outside the BSC, use sealed containers to maintain sample integrity and prevent spillage in case of accidental drops. Preferably, disposable components should be used throughout the procedure to mitigate the possibility of cross-contamination. In cases where disposables are not feasible, ensure all equipment and materials are sterile prior to use. Proper waste management is crucial; all used disposable components should be discarded as biohazard waste. Autoclave reusable materials before reuse to ensure proper sterilization and containment of potentially hazardous materials. Adhering to these precautions not only safeguards sample integrity but also minimizes the risk of operator exposure to microbial pathogens.

1. Preparation of meat samples

1. Acquiring and processing meat samples
   1. Fresh meat
      1. Acquire ground chicken from the fresh meat department of local retailers. Transfer all samples to storage at 4 °C and process within 24 h after receipt. Aseptically divide meat into 25 g samples.
      2. Vacuum seal and irradiate the sample. Here, Texas A&M AgriLife National Center for Electron Beam Research irradiated meat subjected to a dose of ~25 kGy.
         NOTE: While irradiation was used as a control measure in this study to ensure the elimination of background microflora, it is not a prerequisite for the protocol in practical applications as shown by the non-irradiated ready-to-cook products used in the subsequent section. In field settings, alternative methods such as selective media or specificity of molecular diagnostics can address potential interference from non-target microorganisms.
   2. Ready-to-cook chicken products
      1. Acquire ready-to-cook chicken products from the frozen food section of local retailers. Aseptically divide into 25 g samples.
      2. Collect samples from the center of the individual pieces to ensure all ingredients (e.g., breading and cheese) are included.
2. Media preparation
   1. Prepare Buffered Peptone Water (BPW) by dissolving 25 g of BPW powder in 1 L of nanopure H₂O.
   2. Prepare brain heart infusion (BHI) plates. To do this, dissolve 37 g of BHI powder in 1 L of nanopure H₂O and add 15 g of agar into BHI solution. Sterilize all media by autoclaving at 121 °C for 15 min. Pour 20 to 25 mL of media into Petri Dishes with Clear Lid (100 mm x 15mm).
      NOTE: Its best to pour the plates within a biological safety cabinet to maintain aseptic conditions.

2. Cell culture

1. Prepare the initial culture by streaking Salmonella enterica serovar Typhimurium ATCC 14028 on a BHI agar plate and incubate at 37 °C overnight.
2. Prepare overnight cultures by inoculating 25 mL of BHI broth with one colony of freshly grown Salmonella. Aerobically grow cultures overnight at 37 °C with shaking at 100 rpm.

3. Inoculation of poultry samples

1. Culture dilution and plating
   1. Prepare a series of 10-fold dilutions of the overnight culture in BPW to achieve final concentrations of approximately 1 x 10⁸ to 1 x 10¹ CFU/mL. Assume the concentration of the Salmonella overnight culture is 1 x 10⁹ CFU/mL.
   2. Transfer 0.5 mL of the overnight culture into 4.5 mL of BPW, mix, then transfer 0.5 mL of the dilution into 4.5 mL of BPW for each additional dilution.
      1. Spread 10 µL of the 1 x 10³ CFU/mL dilution on a BHI agar plate in triplicate for cell enumeration to calculate the overnight culture concentration.
2. Inoculation of meat samples
   1. Aseptically transfer 25 g of irradiated ground chicken into a sterile stomacher bag in duplicate. The bag is 7.5 x 12 inches and is labeled as 1.63 L. The bag contains a filter partition with a hole diameter of 330 µm, and there are 285 per square cm.
   2. Inoculate each sample with 1 mL of the target concentration dilution. For example, add 1 mL of culture from the 1 x 10³ CFU/mL dilution to achieve a level of contamination of approximately 1,000 cells/25 g chicken. Gently distribute the liquid inoculum over the surface of the chicken samples using a sterile cell spreader and allow it to stand for 1 h at 4 °C.
   3. Prepare negative control samples by adding 1 mL of sterile BPW.

4. Sample processing

1. Add 225 mL of BPW to each sample. The ratio of volume to media was selected to align with FSIS MLG 4.15⁴.
2. Homogenize the samples using the Stomacher⁷ at normal speed and a 120 s duration.
3. Centrifugation and resuspension
   1. Carefully remove the liquid from the filtered side of the bag using a 50 mL pipette. Split the liquid into two sterile centrifuge bottles.
   2. Balance the bottles with sterile BPW to ensure equal weights. Centrifuge the samples at 10,000 x g for 10 min. Discard the supernatant. Resuspend the cell pellet in 3 mL of BPW broth with a sterile spatula.
   3. Add an additional 27 mL of BPW broth and mix thoroughly by stirring with a spatula. Combine the contents of both centrifuge bottles into one bottle for each sample.

5. MPN block set-up

NOTE: Table 1 depicts a schematic of the dilutions in a 48-well block.

1. Add 3 mL of the resuspended sample to each well in column 1 of the 48-well block (8 replicates).
2. Prepare a series of 10-fold dilutions across columns 1-6 within the block using an eight-channel pipette.
3. Add 0.3 mL of sample into 2.7 mL of BPW pipette to mix. Repeat for each dilution. Incubate blocks overnight (~18 h) at 37 °C with shaking at ~100 rpm.

6. Plating and enumeration

1. Modified drop plate enumeration
   1. Plate 7 µL of overnight grown sample of each dilution in a 4 x 6 grid on an agar plate using a multichannel pipette (Figure 2). Using a 4 x 6 grid on two plates better accommodates 8 samples, as opposed to the typical 6 x 6 grid of droplets⁸.
   2. Allow plates to air dry for 10 min before incubation. Incubate the agar plates overnight (~18-24 h) at 37 °C. After incubation, count the number of colonies on each plate.

7. qPCR detection of Salmonella

1. DNA extraction using a commercial kit
   1. Mix cultures in the 48-well block by pipetting up and down several times. Pipette 200 µL of each culture into a 96-well PCR plate.
   2. Seal and then centrifuge the plate at 6,600 x g for 10 min. Remove the supernatant and add 20 µL of the kit reagent to the pellet.
   3. Resuspend the pellet by pipetting up and down. Seal and heat the plate at 99 °C for 10 min, followed by cooling to 20 °C.
   4. Centrifuge again at 6,600 x g for 10 min. Use 2 µL of the supernatant for qPCR analysis.
2. Plate setup
   1. Prepare the qPCR reaction mixture according to established protocol⁹ as follows: 10 µL of 2x Master Mix; 0.4 µL of each primer and probe (10 µM working solution): invA forward: 5'-GTTGAGGATGTTATTCGCAAAGG-3', invA reverse: 5'-GGAGGCTTCCGGGTCAAG-3', invA probe: 5'-CCGTCAGACCTCTGGCAGTACCTTCCTC-3' labeled with the Cal Fluor Orange 560 fluorenes dye; 0.2 µL of internal amplification control (IAC) template (6 x 10⁴ copy/µL)⁹; 0.4 µL of each IAC primer and probe (10 µM): IAC forward: 5'-GGCGCGCCTAACACATCT-3', IAC reverse: 5'-TGGAAGCAATGCCAAATGTGTA-3', IAC probe: 5'-TTACAACGGGAGAAGACAATGCCACCA-3' labeled with TAMRA dye. Adjust volume with ddH₂O to 20 µL total.
   2. Perform real-time PCR with the following cycling conditions⁹: 95 °C for 10 min (initial denaturation of DNA and activation of hot-start polymerase), 40 cycles of 95 °C for 15 s and 60 °C for 1 min, use default Ct settings to export results for analysis.

8. Detection using 3M MDS assay

1. Follow the molecular detection assay Salmonella kit protocol. Mix cultures in the 48-well block by pipetting up and down several times. Pipette 20 µL of each sample into the kit-provided lysis tube.
2. Heat the samples at 100 °C for 15 min. The solution will turn from pink to yellow. Incubate the samples for 10 min at room temperature. The solution will change from yellow to pink.
3. Transfer 20 µL of lysate into a reagent tube and load the reagent tubes into the holder.
4. Add the holder to the MDS instrument and configure the software to communicate the information about the kit and the sample. The instrument requires each well to be labeled with the lot number for the assay and a sample name. Run the MDS software and export the report.

9. Data analysis

1. Classification of positive and negative results.
   1. For 4 x 6 drop plating, assess spots on agar plates with at least 1 colony as positive and spots on agar plates with no growth as negative.
   2. For qPCR, assess wells that have a Ct less than or equal to 30 as positive and wells that have a Ct greater than 30 as negative.
   3. For MDS, use the results from the MDS system, reported as positive or negative.
2. MPN calculation
   1. Analyze annotated positives and negatives using the simple maximum probability resolution (SMPR) method previously described⁶ or alternative verified MPN calculators.¹⁰

Results

Irradiated meat
In regression analysis, a slope of 1 indicates that for every unit increase in the independent variable (x-axis), the dependent variable (y-axis) increases by exactly 1 unit. This suggests a proportional relationship between the two variables, meaning that the change in the dependent variable mirrors the change in the independent variable. An intercept of 0 means that when the independent variable is 0, the dependent variable is also 0. This suggests that there is no fixed offset or...

Discussion

Significance of the protocol
Salmonella remains a major concern in food safety, particularly within poultry products, which are often implicated in foodborne illness outbreaks^13,14. As a leading cause of bacterial foodborne illness in the United States, reliable methods for detecting Salmonella in both fresh and ready-to-cook poultry products are critical to ensuring food safety¹⁵. The ability to qu...

Materials

Name	Company	Catalog Number	Comments
48 deep well block 4.6ml	Fisher Scientific International, Inc	NC1964628
Agar - Solidifying Agent (Difco)	Becton, Dickinson and Company (BD)	281230
Analytical Balance	Mettler Toledo	JL602-G/L	Equipment
Analytical Balance	Mettler Toledo	AB54-S	Equipment
Autoclave - Amsco Lab250, Laboratory Steam Sterilizer	Steris plc	LV-250	Equipment
Biological Safety Cabinet, Type A2, Purifier Logic+	Labconco Corporation	302411101	Equipment
Brain Heart Infusion (BHI) Broth	Becton, Dickinson and Company (BD)	237500
Buffered Peptone Water	Bio-Rad Laboratories Inc.	3564684
Cell Spreader - L-shaped	VWR	76208-438
Centrifuge Microcentrifuge 5424	Eppendorf	5424	Equipment
Centrifuge, Avanti J-25	Beckman Coulter, Inc.		Equipment
DNA Extraction - PreMan Ultra Sample Preparation Reagent	Thermo Fisher Scientific Inc.	4318930
Ground Chicken	Local retailers
IAC forward  primer: 5'-GGCGCGCCTAACACATCT-3'	Integrated DNA Technologies
IAC probe: 5'-TTACAACGGGAGAAGACAATGC CACCA-3' labeled with 5' TAMRA/3' BHQ-2	Biosearch Technologies
IAC reverse primer: 5'-TGGAAGCAATGCCAAATGTGTA-3'	Integrated DNA Technologies
Incubator - Inova 4230 incubator shaker	New Brunswick Scientific	4230	Equipment
Inoculating Loop - Combi Loop  10µL and 1µL	Fisher Scientific International, Inc	22-363-602
invA forward primer: 5'-GTTGAGGATGTTATTCGCAAAG G-3'	Integrated DNA Technologies
invA probe: 5'-CCGTCAGACCTCTGGCAGTAC CTTCCTC-3' labeled with 5' Cal Fluor Orange 560/3' BHQ-1	Biosearch Technologies
invA reverse primer: 5'-GGAGGCTTCCGGGTCAAG-3'	Integrated DNA Technologies
Irradiation Treatment	Texas A&M Agrilife Research National Center for Electron Beam Research		Service
Luria Bertani (LB) Broth	Becton, Dickinson and Company (BD)	244620
Manual pipette Pipet-Lite LTS Pipette L-1000XLS+	Mettler Toledo	17014382	Equipment
Manual pipette Pipet-Lite LTS Pipette L-100XLS+	Mettler Toledo	17014384	Equipment
Manual pipette Pipet-Lite LTS Pipette L-10XLS+	Mettler Toledo	17014388	Equipment
Manual pipette Pipet-Lite LTS Pipette L-200XLS+	Mettler Toledo	17014391	Equipment
Manual pipette Pipet-Lite LTS Pipette L-20XLS+	Mettler Toledo	17014392	Equipment
Manual pipette Pipet-Lite Multi Pipette L8-200XLS+	Mettler Toledo	17013805	Equipment
Manual pipette Pipet-Lite Multi Pipette L8-20XLS+	Mettler Toledo	17013803	Equipment
Media Storage Bottle -PYREX 1L Square Glass  Bottle, with GL45 Screw Cap	Corning Inc.	1396-1L	Equipment
Media Storage Bottle -PYREX 2L Round Wide Mouth Bottle, with GLS80 Screw Cap	Corning Inc.	1397-2L	Equipment
Microtiter plate, 96 well plate, flat bottom, polystyrene, 0.34cm2, sterile, 108/cs	MilliporeSigma	Z707902
Mixer - Vortex Genie 2	Scientific Industries Inc.	SI-0236	Equipment
Molecular Detection Assay 2-Salmonella kit	Neogen	MDA2SAL96
Molecular Detection Instrument	Neogen	MDS100	Equipment
Motorized pipette controller, PIPETBOY2	INTEGRA Biosciences Corp.	155019	Equipment
PCR Mastermix 2× TaqMan Gene Expression	Thermo Fisher Scientific Inc.	4369542
Petri Dish Rotator -  bioWORLD Inoculation Turntable	Fisher Scientific International, Inc	3489E20	Equipment
Petri Dishes with Clear Lid (100 mm x 15mm)	Fisher Scientific International, Inc	FB0875713
Pipette Tips GP LTS 1000µL S 768A/8	Mettler Toledo	30389273
Pipette Tips GP LTS 20µL 960A/10	Mettler Toledo	30389270
Pipette Tips GP LTS 200µL F 960A/10	Mettler Toledo	30389276
Ready to cook chicken products	Local retailers
Reagent Reservoir, 25 mL sterile reservoir used with multichannel pipettors	Thermo Fisher Scientific Inc.	8093-11
Realtime PCR - 7500 Real-Time PCR system	(Applied Biosystems, Foster City, CA)	2750036476	Equipment
Serological Pipettes, Nunc Serological Pipettes (10 mL)	Thermo Fisher Scientific Inc.	170356N
Serological Pipettes, Nunc Serological Pipettes (2 mL)	Thermo Fisher Scientific Inc.	170372N
Serological Pipettes, Nunc Serological Pipettes (25 mL)	Thermo Fisher Scientific Inc.	170357N
Serological Pipettes, Nunc Serological Pipettes (50 mL)	Thermo Fisher Scientific Inc.	170376N
Spreader - Fisherbrand L-Shaped Cell Spreaders	Fisher Scientific International, Inc	14-665-230
Stomacher bag, Nasco Whirl-Pak Write-On Homogenizer Blender Filter Bags	Thermo Fisher Scientific Inc.	01-812
Stomacher 80 Biomaster Lab Blender	Seward	30010019	Equipment
Thermocycler (GeneAmp PCR system 9700)	Applied Biosystems	535028293	Equipment
Water Filtration - Elga Veolia Purelab Flex	Elga LabWater	PF2XXXXM1-US	Equipment
Whirlpak bags 1.63L	VWR	11216-777